The field of the invention is neuronal cell death.
Neuronal cell death can occur as a result of a variety of conditions including traumatic injury, ischemia, degenerative disease (e.g., Parkinson""s disease, ALS, or SMA), or as a normal part of tissue development and maintenance.
We have previously discovered the NAIP and the IAP proteins (see U.S. Ser. No. 08/511,485 now U.S. Pat. No. 5,919,912; Ser. No. 08/576,956 now U.S. Pat. No. 6,156,535; and 60/017,354, incorporated by reference). These proteins are involved in the control of apoptosis.
Developmental cell death, or apoptosis, is a naturally occurring process thought to play a critical role in establishing appropriate neuronal connections in the developing central nervous system (CNS). Apoptosis is characterized morphologically by condensation of the chromatin followed by shrinkage of the cell body. Biochemically, the hallmark of apoptosis is the degradation of nuclear DNA into oligonucleosomal fragments (multiples of 180 basepairs) mediated by a Ca2+/Mg2+-dependent endonuclease. DNA laddering precedes cell death and may be a key event leading to death. In keeping with this proposal, agents which inhibit DNA fragmentation prevent apoptosis, whereas morphology indicative of apoptosis is produced by enzymes that digest nuclear DNA. Apoptosis is often dependent on RNA and protein synthesis within the dying cell suggesting the activation of a cell death pathway. The best defined genetic pathway of cell death is in the nematode Caenorhabditis where both effector (ced-3 and ced-4) and repressor (ced-9) genes have been isolated. Similar genes have been identified in mammals. One such example is the proto-oncogene bcl-2 which is thought to be the mammalian homolog of ced-9. Overexpression of Bcl-2 has been shown to render neurons resistant to the damaging effects of a wide variety of noxious treatments. However, the very low levels of Bcl-2 detected in adult brain suggest that other proteins may play a more important role in preventing apoptosis in the mature CNS.
Spinal muscular atrophy (SMA) is a hereditary neurodegenerative disorder characterized by a severe depletion of motor neurons in the spinal cord and brain stem. Many of the motor neurons observed at autopsy in SMA spinal cords display such features as chromatolysis which are consistent with the apoptosis. During maturation of the spinal cord, as many as 50% of motor neurons undergo apoptosis. This has led to the suggestion that a genetic defect in a neuronal apoptotic pathway may be responsible for motor neuron depletion in SMA. Recently two candidate genes, i.e., survival motor neuron (smn) and neuronal apoptosis inhibitory protein (naip), have been identified. The product of the naip gene was termed neuronal apoptotic inhibitory protein (NAIP) because of sequence homology with two baculoviral proteins (Cp-IAP and Op-IAP) that block virally induced apoptosis.
Parkinson""s disease (PD) is a progressive neurodegenerative disorder characterized by a loss of nigrostriatal neurons which results in a severe depletion of dopamine (DA) levels in the basal ganglia. Rats which have sustained unilateral lesions of the nigrostriatal pathway produced by the catecholamine-specific neurotoxin, 6-hydroxydopamine (6-OHDA) serve as an animal model of PD. Unilateral injection of 6-OHDA into the medial forebrain bundle or the substantia nigra pars compact results in a rapid degeneration of the nigrostriatal pathway. However, injection of 6-OHDA into the striatum produces a progressive degeneration ( greater than 1 week) of the nigrostriatal pathway which is believed to more closely resemble the natural pathology of PD (Sauer and Oertel, 1994). No good treatment for the prevention of PD degeneration currently exists.
Epilepsy is characterized by brain seizures and often results in neural cell death. It has been observed that previously kindled rats, i.e., those rendered xe2x80x9cepilepticxe2x80x9d via daily application of low intensity electrical stimulation, show considerably less brain damage from kainic-acid induced status epilepticus as compared to naive rats. Status epilepticus (SE) is characterized by continuous electrographic and/or behavioral seizures that occur unabated for greater than 30 minutes. In naive, nonkindled rats, this condition ultimately results in severe neuronal damage in several brain regions. Despite experiencing a form of SE as severe as that observed in a naive rat, the kindled xe2x80x9cepilepticxe2x80x9d rats show only minimal neuronal loss. Determining the mechanisms responsible for this experience-induced neuroprotection could provide novel approaches to the amelioration of brain damage resulting not only from SE, but also from other neurological traumas such as stroke.
Ischemia results when blood flow to the CNS is interrupted. This is frequently what happens following traumatic injury and stroke. Cell death often results. If this interruption of blood flow effects a large area of the CNS, or lasts for a long period of time, death due to loss of neurological function required for viability occurs. However, if blood flow to the CNS is transiently interrupted and recirculation is established within minutes, only certain neurons in the brain will die.
The best experimental model partial neuronal death due to ischemia is the 4-vessel occlusion model. In this global model of cerebral ischemia, neurons in neocortical layers 3, 5, and 6xe2x80x2, small- and medium-sized striatal neurons; and hippocampal CA1 pyramidal neurons are among the most vulnerable (Pulsinelli et al., Ann. Neurol. 11:491-498, 1982). By contrast, cholinergic interneurons in the striatum and CA3 pyramidal neurons in the hippocampus are more resistant to the damaging effects of transient global ischemia (Francis and Pulsinelli, Brain Res. 243:271-278, 1982).
We have discovered that increased levels of NAIP and IAP polypeptides used provide neuroprotection and allow neural regeneration.
We have found that increased NAIP levels correlate with neuronal survival under a variety of conditions which normally result in neuronal cell death. Furthermore, increased NAIP and IAP levels allow axonal regrowth after axotomy.
Taken together, these findings indicate that NAIP and the IAPs play a key role in conferring resistance to ischemic damage and neural degeneration, and allowing neural repair. Accordingly, our discovery provides both methods for the prevention of neural damage and neural repair and methods by which to screen for neuroprotective and neuroregenerative compounds.
The invention may be summarized as follows.
In the two principle aspects, the invention provides a method for inhibiting death of a cell of the nervous system and/or enhancing neural regeneration. The methods include increasing the biological activity (e.g., levels or neuroprotective effects as described herein) of a polypeptide selected from the group consisting of the NAIP or an TAP. This increasing in cells exposed or likely to be exposed to ischemic conditions is part of the invention if it is sufficient to produce a 20% or greater increase in the likelihood that a cell will survive following an event which normally causes at least a degree of nerve cell death. In some embodiments, the polypeptide is mammalian NAIP, HIAP, HIAP2, or XIAP. Most preferably the polypeptide is NAIP. In one preferred embodiment the polypeptide is a NAIP polypeptide lacking a portion of the carboxy-terminus. In a related embodiment the nerve cell is the CNS, and most preferably, the cell is a neuronal cell known to be susceptible to post-ischemic cell death.
In another embodiment the increasing is by administration of a transgene encoding the NAIP or TAP polypeptide in an expressible genetic construct. The transgene may be in a construct which includes various types of promoters, e.g., a constitutive promoter, a neurofilament promoter, or a regulatable promoter.
The transgene may be in a viral vector, e.g., an adenovirus vector, a herpes virus vector, or a polio virus vector. In preferred embodiments, the transgene encodes a NAIP or an TAP including an amino acid sequence substantially identical to at least one of the amino acid sequences described in the references provided herein; the transgene is administered to the mammal in the region of the ischemic event (preferably intracranially) the transgene is included in a viral vector (for example, a herpesvirus, adenovirus, adeno-associated virus or poliovirus vector); and the ischemia is due to traumatic injury, stroke, myocardial infarction, or mini-stroke.
In another embodiment, the invention includes a method of treating a mammal who has experienced or is at an increased risk for experiencing an ischemic event, neurodegeneration, or axotomy (for example, a stroke Parkinson""s disease or surgical injury, respectively), involving administering to the mammal a NAIP or an IAP in an amount sufficient to inhibit cell death and/or allow regeneration, and further features a therapeutic composition having as an active ingredient a NAIP or an IAP, formulated in a physiologically-acceptable carrier.
In yet other embodiments, the increasing may be by directly administering a NAIP or IAP (e.g., HIAP1, HIAP2, or XIAP), or by administering a molecule found to increase NAIP or IAP-biological activity (e.g., K252a-like alkaloid other than K252a, an ATP analog, or a staurosporine-like compound).
The methods may be used to treat patients diagnosed as having had an ischemic event, suspected to have had or diagnosed as having a predisposition to an ischemia event, a degenerative disease, or axon damage. In the therapeutic methods of the invention the therapeutic molecule is most preferably provided as soon as the predisposition to ischemia, degeneration, or damage has first been detected. Nonetheless, the therapy may be provided up to 72 hours after the ischemic effects event. In the case of degeneration or damage, beneficial effects may be obtained a considerable time after the first detection of the condition.
In some embodiments of the assay the cell is a neuronal cell or a related cell such as a glial cell, the cell is in a mammal (e.g., a mouse), or is in culture.
In two other related aspects, the invention features methods of identifying NAIP or an IAP modulatory compounds or compounds which mimic the neuroprotective or neuroregenerative effects. These methods may be used for detection of therapeutics for neuroprotection and nerve regeneration. The first method involves the identification of modulatory compounds that are capable of increasing the expression or stability of a NAIP or an IAP gene mRNAs or polypeptides, involving (a) providing a cell expressing the NAIP or an IAP; and (b) contacting the cell with a candidate compound, an increase in NAIP or an IAP mRNA or protein expression following contact with the candidate compound identifying a modulatory compound. The second method involves the identification of modulatory compounds which are capable of increasing NAIP or an IAP biological activity, involving (a) providing a cell expressing a NAIP or an IAP; and (b) contacting the cell with a candidate compound, a decrease in NAIP or an IAP expression following contact with the candidate compound identifying a modulatory compound. Preferably, the method also includes monitoring the level of neural cell death in the presence of the compound by contacting a cell (preferably a neuronal cell in culture) susceptible to cell death with the compound. A decrease in cell death indicates a compound with increased promise as neuroprotective or neuroregenerative compound. As one skilled in the art can appreciate, increases in NAIP or IAP biological activity can also be done using cell-free systems and solid state assay systems known to one skilled in the art and readily adaptable for this purpose.
In preferred embodiments of both methods, the NAIP or an IAP gene encodes or the NAIP or an IAP and includes an amino acid sequence that is substantially identical to one of the amino acid sequences described in the references provided herein (including NAIP having a carboxy terminal truncation); the candidate compound may be chosen from a compound bank, or more preferably is a K252a-like compound, a staurosporine-like compound, an ATP analog, or a growth factor-like compound (e.g., a neurotrophic compound).
In a related aspect, the invention features a method of treating a mammal who has experienced or is at an increased risk for experiencing an ischemic event, involving administering to the patient a modulatory compound (for example, identified according to the above methods) in an amount effective to reduce the cell deaths in the mammal. Preferably, the modulatory compound acts by increasing NAIP or an IAP gene expression.
Kits for carrying out the above methods are also included in the invention. Such kits preferably include a substantially pure antibody that specifically recognizes and binds a NAIP or an IAP, and may also include means for detecting and quantitating antibody binding. Alternatively, the kit may include all or a fragment of a NAIP or an IAP nucleic acid sequence useful for hybridization purposes, and may also include means for detecting and quantitating NAIP or an IAP RNA hybridization. In yet another alternative the kit may include a cell system or cell-free for monitoring NAIP or IAP expression. Kits may also include instructions sufficient to allow determination of the detection of a neuroprotective or neuroregenerative compound.
By xe2x80x9cNAIP or an IAPxe2x80x9d is meant an amino acid sequence which has homology to baculovirus inhibitors of apoptosis. For example, NAIP, truncated NAIP, HIAP1, HIAP2 and XIAP are specifically included (see U.S. Ser. No. 08/511,485, filed Aug. 4, 1995 now U.S. Pat. No. 5,919,912; Ser. No. 08/576,956, filed Dec. 22, 1995 now U.S. Pat. No. 6,156,535; and PCT/IB97/00142, filed Jan. 17, 1997). Preferably, such a polypeptide has an amino acid sequence which is at least 45%, preferably 60%, and most preferably 85% or even 95% identical to at least one of the amino acid sequences of the NAIP, truncated NAIP, HIAP1, HIAP2, or XIAP described in the references provided herein.
By a xe2x80x9csubstantially identicalxe2x80x9d polypeptide sequence is meant an amino acid sequence which differs only by conservative amino acid substitutions, for example, substitution of one amino acid for another of the same class (e.g., valine for glycine, arginine for lysine, etc.) or by one or more non-conservative substitutions, deletions, or insertions located at positions of the amino acid sequence which do not destroy the function of the polypeptide (assayed, e.g., as described herein).
Preferably, such a sequence is at least 85%, more preferably 90%, and most preferably 95% identical at the amino acid level to the sequence described in the references provided herein. For polypeptides, the length of comparison sequences will generally be at least 15 amino acids, preferably at least amino acids, more preferably at least 25 amino acids, and most preferably at least 35 amino acids.
Identity is typically measured using sequence analysis software (e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705). Such software matches similar sequences by assigning degrees of identity to various substitutions, deletions, substitutions, and other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine.
By xe2x80x9cproteinxe2x80x9d or xe2x80x9cpolypeptidexe2x80x9d is meant any chain of amino acids, regardless of length or post-translational modification (e.g., glycosylation or phosphorylation).
By xe2x80x9csubstantially purexe2x80x9d is meant a preparation which is at least 60% by weight (dry weight) the compound of interest, e.g., NAIP or an IAP or NAIP or an IAP-specific antibody. Preferably the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight the compound of interest. Purity can be measured by any appropriate method, e.g., column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.
By xe2x80x9cincreasing biological activityxe2x80x9d is meant increasing transcription of the relevant gene, translation of the relevant mRNA, increasing stability of the relevant RNA, modification of the polypeptide to enhance stability, enhancement of neuroprotective or neuroregenerative activity, e.g., in an assay provided herein, and administration of compounds which stabilize the polypeptide.
By xe2x80x9cpurified DNAxe2x80x9d is meant DNA that is not immediately contiguous with both of the coding sequences with which it is immediately contiguous (one on the 5xe2x80x2 end and one on the 3xe2x80x2 end) in the naturally-occurring genome of the organism from which it is derived. The term therefore includes, for example, a recombinant DNA which is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (e.g., a cDNA or a genomic DNA fragment produced by PCR or restriction endonuclease treatment) independent of other sequences. It also includes a recombinant DNA which is part of a hybrid gene encoding additional polypeptide sequence.
By a xe2x80x9csubstantially identicalxe2x80x9d nucleic acid is meant a nucleic acid sequence which encodes a polypeptide differing only by conservative amino acid substitutions, for example, substitution of one amino acid for another of the same class (e.g., valine for glycine, arginine for lysine, etc.) or by one or more non-conservative substitutions, deletions, or insertions located at positions of the amino acid sequence which do not destroy the function of the polypeptide (assayed, e.g., as described herein). Preferably, the encoded sequence is at least 45%, more preferably 60%, and most preferably 85% identical at the amino acid level to at least one of the NAIP or IAP sequences provided by the references herein. If nucleic acid sequences are compared a xe2x80x9csubstantially identicalxe2x80x9d nucleic acid sequence is one which is at least 85%, more preferably 90%, and most preferably 95% identical to the NAIP or IAP sequences referenced herein. The length of nucleic acid sequence comparison will generally be at least 50 nucleotides, preferably at least 60 nucleotides, more preferably at least 75 nucleotides, and most preferably 110 nucleotides. Again, homology is typically measured using sequence analysis software (e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705).
By xe2x80x9cpositioned for expressionxe2x80x9d is meant that the DNA molecule is positioned adjacent to a DNA sequence which directs transcription and translation of the sequence (i.e., facilitates the production of NAIP or an IAP protein).
By xe2x80x9cpurified antibodyxe2x80x9d is meant antibody which is at least 60%, by weight, free from the proteins and naturally-occurring organic molecules with which it is naturally associated. Preferably, the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight, antibody.
By xe2x80x9cspecifically bindsxe2x80x9d is meant an antibody which recognizes and binds a NAIP or an IAP but which does not substantially recognize and bind other molecules in a sample (e.g., a biological sample) which naturally includes NAIP or an IAP. An antibody which xe2x80x9cspecifically bindsxe2x80x9d NAIP or an IAP is sufficient to detect a NAIP or an IAP protein product in such a biological sample using one or more of the standard immunological techniques available to those in the art (for example, Western blotting or immunoprecipitation).
By xe2x80x9cischemiaxe2x80x9d is meant any disruption of blood flow that causes cellular anoxia more rapidly than normal and which leads to cell death. Most ischemia is the result of a partial or complete lack of blood flow. Ischemia according to the invention is preferably ischemia of cells of the CNS, most preferably neuronal cells of the CNS.
By xe2x80x9crelative to a wild-type samplexe2x80x9d is meant either (a) relative to an equivalent tissue sample from a non-ischemic area of an animal or (b) relative to an non-ischemic cell in culture.
By xe2x80x9cimmunological methodsxe2x80x9d is meant any assay involving antibody-based detection techniques including, without limitation, Western blotting, immunoprecipitation, and direct and competitive ELISA and RIA techniques.
By xe2x80x9cmeans for detectingxe2x80x9d is meant any one or a series of components that sufficiently indicate a detection event of interest. Such means involve at least one label that may be assayed or observed, including, without limitation, radioactive, fluorescent, and chemiluminescent labels.
By xe2x80x9cNAIP or an IAP RNAxe2x80x9d is meant messenger RNA transcribed from a NAIP or an IAP DNA sequence.
By xe2x80x9chybridization techniquesxe2x80x9d is meant any detection assay involving specific interactions (based on complementarity) between nucleic acid strands, including DNA-DNA, RNA-RNA, and DNA-RNA interactions. Such hybridization techniques may, if desired, include a PCR amplification step.
By xe2x80x9ctransgenexe2x80x9d is meant a nucleic acid sequence which is inserted by artifice into a cell and becomes a part of the genome of that cell and its progeny. Such a transgene may be partly or entirely heterologous to the cell.
By xe2x80x9cmodulatory compoundxe2x80x9d, as used herein, is meant any compound capable of either increasing NAIP or an IAP expression (i.e., at the level of transcription, translation, or post-translation) or increasing NAIP or an IAP protein activity (i.e., by stabilizing the active form of the protein or by otherwise decreasing the amount of apoptosis in a cell type susceptible to apoptosis).
By xe2x80x9cischemic cell deathxe2x80x9d is meant, loss of cell viability following a decrease or blockage in blood flow to the affected cell.
By xe2x80x9cinhibiting cell deathxe2x80x9d is meant at least a 10% preferably or 20%, increase in the likelihood that a cell will survive following an event which normally causes cell death (relative to an untreated control cell). Preferably, the cells being compared are neural cells normally susceptible to ischemic cell death, neurodegeneration, or axotomy. Preferably, the decrease in the likelihood that a cell will die is 80%, more preferably 2-fold, most preferably, 5-fold.
Abbreviations used herein are as follows: oculomotor nucleus (3); oculomotor nerve root (3nr); facial nucleus (7); vestibulocochlear nerve (8vn); nucleus vagus (10); hypoglossal nucleus (12); cervical 5 (C5); Clarke""s column (CC); cuneate nucleus (Cu); dorsal hypothalamal area (DA); dentate gyrus (DG); dorsal medial spinal trigeminal nucleus (DMSP5); endopeduncular nucleus (EP); granule cell layer (G); globus pallidus (GP); gracile nucleus (Gr); Edinger-Westphal nucleus (EW); horizontal diagonal band (HDB); interposed cerebellar nucleus (int);stratum lucunosum (L); lateral cerebellar nucleus (lat); locus coeruleus (LC); lateral hypothalamal area (LH); lateral habenular nuclei (Lhb); stratum moleculare (M); mesencephalic trigeminal nucleus (Me5); medial habenular nuclei (Mhb); motor trigeminal nucleus (Mo5); stratum oriens (O); pyramidal cell layer (P) (FIG. 2); Purkinje cell layer (FIG. 11); pontine nucleus (Pn); principal trigeminal sensory nucleus (Pr5);stratum radiatum (R); red nucleus, magnocellular part (RMC); red nucleus, parvocellular part (RPC); rostral periolivary area (RPO); sacral spinal cord (S); substantia nigra pars compact (SNC); substantia nigra pars reticulata (SNR); substantia nigra pars lateralis (SNL); spinal trigeminal nucleus, oral part (SPO5); ventral cochlear nucleus (VC); vertical diagonal band (VDB); vestibular nucleus (Ve); thoracic 2 (T2); and thoracic 6 (T6).
Other features and advantages of the invention will be apparent from the following detailed description thereof, and from the claims.